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Type of publication: Baccalaureate Thesis
Type of document:

Year: 2010

Authors: Haas, Jasmin

Title: Insertion site analysis of EIAV based vectors in mouse model for hemophilia B gene therapy.

Source: Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 40.


Advisor(s):

Klein Dieter

Reviewer(s):
G├╝nzburg Walter

Vetmed Research Units:
VetCore


Abstract:
The investigation of the insertion sites of the EIAV based vectors in mouse model for hemophilia B gene therapy is the theme of this bachelor thesis. EIAV proviral SMART 2Z vector or SMART hFIX vector, containing the factor IX gene or the β-galactosidase (LacZ) gene, were delivered in hFIX-knockout mice inducing a stable and persistent virus integration in the mouse genome guaranteeing a high level of gene expression to the searched factor IX protein, which is important for the coagulation cascade to a correct blood clotting. The genotoxicity of this vector became obvious, when gene therapy treated mice developed hepatocellular tumors in the liver. On the cellular level virus binds to molecular receptors on the surface of the Kupffer cells in the liver, is then absorbed into the cell and its RNA genome is reverse transcribed into dsDNA. This dsDNA enters the nucleus and integrates in the host cell genome. In this bachelor thesis the vector-genome transition is amplified with linear amplification-mediated PCR (LAM-PCR) and then sequenced with the next generation 454-Pyrosequenzing. The data is bioinformatically analyzed with Reference Sequence Gene, retroviral insertion site (RIS) distribution and then the details of gene pathways leading to the development of cancer are investigated with Ingenuity. Basic statistical examination found 62 (SMART2Z) and 205 (SMART hFIX) integrations sites. RIS distribution shows that the virus integrates less randomly with a preference for integration within the first seven chromosomes. In contrast to the results obtained by in the X-SCID1 study, where γ-retrovirus integrates preferably to transcription start sites, the EIAV virus integrates preferably a rate of 70 to 80 % in active genes or 10 kilobases pairs up- or downstream of those. Ingenuity analysis showed the toxic genes in the liver. Network analysis of the healthy liver tissue (both SMART2Z and SMART hFIX) found no tumor-inducing genes, only TGF-α and MAPKA-8 which influence the physiological liver growth. But if TNF-α is active, the activity of TGF-α and MAPKA-8 could lead to abnormal cellular growth. The tumor tissue of the SMART 2Z presented many genes having an impact on the proto-oncogene TP53. Indeed, the sequencing of the hepatocellular tumor-inducing proto-oncogene cMyc-gene in the SMARThFIX tumor liver samples finally proves that the growth of the hepatocellular tumor in the gene therapy liver is primarily based on insertional mutagenesis, triggered by the integration of the EIAV vector into the host cells genome in the vicinity of tumor-inducing oncogenes.


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